Lassa virus (LASV) is a highly prevalent arenavirus in West Africa, where it infects several hundred thousand people annually, resulting in a high number of Lassa fever (LF) cases, a hemorrhagic fever (HF) disease associated with high morbidity and mortality. The impact of LF in human health led to its inclusion on the WHO list of top Priority Diseases in 2017. Moreover, LASV poses a credible threat to biodefense readiness. There are not FDA-licensed LASV vaccines and current anti-arenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Live-attenuated vaccines (LAV) are the most feasible approach to control HF arnaviruses within their endemic regions. The LAV Candid#1 (Can) strain of the arenavirus Junin (JUNV) has been shown to be a safe and effective vaccine to protect humans against Argentine HF caused by JUNV. The central goal of this exploratory R21 grant application is to leverage the established safety record and efficacy in humans of Can, together with our expertise in arenavirus reverse genetics to generate recombinant Can (rCan) expressing LASV antigens (rCan/lasvAg) to test the hypothesis that r3Can/lasvAg represent a novel approach to develop a safe and effective LAV to combat LF. To test our hypothesis we propose to complete the following specific aims (SA): SA 1. Generation and characterization of r3Can expressing LASV antigens (r3Can/lasvAg): We will rescue r3Can/lasvAg expressing NP* or GPC*, or both, LASV antigens known to induce protective immune responses against LASV. NP* contains mutation D478A to prevent its interference with the activity of Can vRNP, whereas GPC* corresponds to a stabilized pre-fusion form that is not incorporated into infectious particles, but predicted to induce a strong neutralizing and protective Ab response against LASV. We will characterize r3Can/lasvAg growth properties in cultured cells and virulence in mice. We will test the hypothesis that r3Can/lasvAg grow to titers compatible for vaccine production but are attenuated in vivo. SA 2. Assess r3Can/lasvAg safety, immunogenicity and protective efficacy in mice: We will assess r3Can/lasvAg safety in IFN- a/bg R KO B6 mice, which are highly susceptible to JUNV. We will evaluate immunogenicity of r3Can/lasvAg by examining both T-cell responses and production of virus-specific antibodies in immunized B6 mice. We will determine r3Can/lasvAg protective efficacy by challenging r3Can/lasvAg immunized B6 mice with rVSV expressing LASV NP and GPC antigens. We will test the hypothesis that r3Can/lasvAg are safe and able to induce LASV antigen-specific protective immunity. SA 3. Determine the stability of r3Can/lasvAg in cultured cells and in vivo: We will test the hypothesis that r3Can/lasvAg are phenotypically and genetically stable during multiplication in cultured cells and in vivo.